Method and device for selective hyperthermic damage of target cells

ABSTRACT

Device and method for selective hyperthermic damage of target cells by means of millimeter wave radiation. Cells in a culture medium are placed in a cell cultureware and millimeter wave radiation is delivered via a waveguide to a predetermined region of exposure of the culture medium. The device and method are safe and efficient for use in clinical and research applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to provisional U.S.patent application Ser. No. 61/950,832, which was filed on Mar. 10,2014.

FIELD OF THE INVENTION

The present invention relates generally to systems and methods forselective hyperthermic damage of target cells within cells in a culturemedium, and more specifically, to a method and device for selectivehyperthermic damage of unwanted cells within a cell population withmillimeter wave radiation.

BACKGROUND OF THE INVENTION

Known methods for targeted elimination of anchorage-dependent cellsgenerally utilize laser technology such as laser ablation and lasermicrodissection. In these methods a high power laser beam (usually froma pulsed UV laser) is used to either sweep over the surface to lethallyilluminate the unwanted cells, or to cut out the cells of interest andphysically separate them from the remaining cells. The shortcoming ofthe laser ablation approach is that a lot of free radicals and productsof oxidation are formed during the ablation process. These aggressivebyproducts harm the desired cells. The microdissection approach is notsterile and requires special consumables. All laser-based methods needspecial and very expensive equipment, precise laser optics adjustmentand in some cases, addition of special light energy absorbing dye to thecell media to intensify the cell damage. The majority of these methodscannot be used with standard microscopes and cannot be performedmanually by an inexperienced operator.

As known in the art, direct use of radiation in the infrared area is notapplicable for selective cell ablation, as this radiation is stronglyabsorbed by glass, plastic and water. Therefore, if an infrared beamwould be applied to glass or plastic bottom of a cell culturewarecontaining cells in a culture medium, it would heat both the culturewarebottom and the medium in the cell cultureware and therefore could not beused for selective local ablation of the cells. Besides, the intenseinfrared radiation is dangerous for vision. Despite not being visible,infrared radiation can still pass through the anterior structures of theeye and reach the retina. Since human eyes are unable to detect infraredradiation, there would be no blink or aversion reflex to protect theeyes from the damage if the eyes are exposed to intense infraredradiation.

Thus, there exists a need for a safe method and device for selectivehyperthermic damage of target cells, specifically for irreversibledamage and ablation of target cells within a cell population, thatovercome the limitations of previously known methods and devices.

A need also exists for a method and device for selective hyperthermicdamage of target cells, which are efficient for use in clinical andresearch applications.

A need further exists for a method and device for selective hyperthermicdamage of target cells which are capable of being implemented with anytype of known standard microscopes and could be performed manually by aninexperienced operator.

SUMMARY OF THE INVENTION

As known in the art, millimeter wave radiation is used in a variety ofapplications, from voice and data transmission and high-resolution radarimaging to body scanning and body imaging.

The present method is based on water absorption of millimeter waveradiation that occurs in both cell medium and cell material. Generally,the method employs millimeter waves that have water penetration depth ofabout 0.4 mm (W-band from 75 to 110 GHz) or less (for F, D and G bands).The millimeter waves are guided to the cell cultureware via a specialwaveguide that provides a localized irradiation spot of a controllablesize. Millimeter waves rapidly heat a narrow layer (about 0.4 mm orless) of a media volume comprising unwanted cells near an externalsurface of a cell cultureware (made from glass or thin plastic) to about58-60° C. The cells locally heated by millimeter wave radiation even fora short time are irreversibly damaged, as mammalian cells in culturemedium or in tissues do not survive if exposed to temperatures exceeding48-50° C. even for a short time interval (less than 1 minute). Accordingto survival data of mammalian cells at elevated temperatures, there areno surviving cells starting at about 48° C. even though the cells areheated for less than 30 seconds. Higher temperatures (about 58-60° C.)sharply shorten the critical exposure time required to kill the cells byheating to less than 2-3 seconds. Such a short time to achieve celldeath at 58-60° C. is attributed to the fast and irreversibledenaturation of cell proteins.

In accordance with the subject application, there is provided animproved method for selective hyperthermic damage of target cells,specifically for irreversible damage of target cells within a cellpopulation, that overcome the limitations of previously known methodsand devices.

Further, in accordance with the subject application, there is providedan improved device for selective hyperthermic damage of target cells,specifically for irreversible damage of target cells within a cellpopulation, that overcome the limitations of previously known methodsand devices.

Still further, in accordance with the subject application, there isprovided a method and device for selective hyperthermic damage of targetcells, which are efficient for use in clinical and researchapplications.

Yet further, in accordance with the subject application, there isprovided a method and device for selective hyperthermic damage of targetcell which are capable of being implemented with any type of knownstandard microscopes and could be performed manually by an inexperiencedoperator.

According to one aspect of the subject application, there is provided amethod for selective hyperthermic damage of target cells. The methodcomprises the steps of providing a millimeter wave radiation; anddelivering the millimeter wave radiation to a predetermined spot of anexternal surface of an associated cell cultureware having cells in aculture medium placed therein, via a waveguide, exposing thereby apredetermined region of exposure of the cells in the culture medium tothe millimeter wave radiation. Radiation power and exposure time of themillimeter wave radiation, and positional relationship of the end faceof the waveguide and of the target cells are selected such as to provideselective hyperthermic damage to the target cells in the predeterminedregion of exposure of the cells in the culture medium.

In an exemplary embodiment, the hyperthermic damage is irreversible.

In one embodiment, the predetermined region of exposure may bevisualized with an infrared sensitive thermal vision camera. In anotherembodiment, the exposure time may be about 2-3 sec, wherein the targetcells in the predetermined region of exposure may be heated to atemperature of about 58-60° C.

In another embodiment, the method may further comprise a step ofvisualizing the predetermined region of exposure with an infraredsensitive thermal vision camera.

In an exemplary embodiment, the exposure time is about 2-3 sec, whereinthe target cells in the predetermined region of exposure are heated to atemperature of about 58-60° C.

In another exemplary embodiment, the method may further comprise a stepof visualizing the target cells in the predetermined region of exposurevia bright-field or fluorescence microscopy.

In another exemplary embodiment, the millimeter wave radiation is in afrequency band of 75 to 110 GHz.

In another embodiment, the predetermined region of exposure has adiameter in the range of from about 0.25 mm to about 1.3 mm.

In an exemplary embodiment, an end face of the waveguide is positionedbelow an associated cell cultureware having cells in a culture mediumplaced therein, in proximity of the predetermined spot, exposing therebythe predetermined region of exposure of the cells in the culture mediumlocated above the predetermined spot, to the millimeter wave radiation.

According to another aspect of the subject application, there isprovided a device for selective hyperthermic damage of target cells thatcomprises a source of millimeter wave radiation, and a waveguide adaptedfor delivering the millimeter wave radiation to a predetermined spot ofan external surface of an associated cell cultureware having cells in aculture medium placed therein, to provide for exposing a predeterminedregion of exposure of the cells in the culture medium to the millimeterwave radiation. An end face of the waveguide is positioned in closeproximity of the predetermined spot. Radiation power and exposure timeof the millimeter wave radiation, and positional relationship of thewaveguide end face and the target cells are selected such as to provideselective hyperthermic damage to the target cells in the predeterminedregion of exposure of the cells in the culture medium.

In one embodiment, the device may further comprise a visualizing moduleadapted for visualizing the cells in the culture medium placed in theassociated cell cultureware. The visualizing module may comprise amicroscope, wherein the associated cell cultureware having cells in theculture medium placed therein is positioned on a microscope stage. Inone embodiment, the microscope may be motorized. In a specificembodiment, the microscope may be a fluorescent or a bright-fieldmicroscope.

In another embodiment, the microscope is an upright microscope, andwherein the end face of the waveguide is positioned below a bottom of anassociated cell cultureware having cells in the culture medium placedtherein, exposing thereby the predetermined region of exposure of thecells in the culture medium located above the predetermined spot, to themillimeter wave radiation. The waveguide may comprise a first portionpositioned horizontally relative to the microscope stage and a secondportion positioned upright relative to the microscope stage, wherein thesecond portion is connected with the first portion via a 90 degreeE-plane waveguide bend.

In an exemplary embodiment, the visualizing module may further compriseat least one of: a long working distance objective, a CCD camera forimage capture, an infra-red camera for visualization on a monitor and amotorized stage to hold and move the associated cell cultureware withthe cells in the culture medium placed therein.

Still other objects and aspects of the present invention will becomereadily apparent to those skilled in this art from the followingdescription wherein there are shown and described preferred embodimentsof this invention, simply by way of illustration of the best modessuited for to carry out the invention. As it will be realized by thoseskilled in the art, the invention is capable of other differentembodiments and its several details are capable of modifications invarious obvious aspects all without departing from the scope of thesubject application. Accordingly, the drawings and description will beregarded as illustrative in nature and not as restrictive.

BRIEF DESCRIPTION OF DRAWINGS

For a more complete understanding of the present invention and theadvantages thereof, reference is now made to the following descriptiontaken in conjunction with the accompanying drawings, in which:

FIG. 1 is a block diagram of one embodiment of a device for selectivehyperthermic damage of target cells in accordance with the subjectapplication.

FIG. 2 is a block diagram of another embodiment of a device forselective hyperthermic damage of target cells in accordance with thesubject application.

FIG. 3 illustrates a calibration process in accordance with the subjectapplication.

FIG. 4 shows the effect of 94 GHz irradiation (heating to 60° C.) onU2OS cells expressing fusion protein of α-tubulin and green fluorescentprotein (GFP-tubulin).

FIG. 5a shows the effect of 94 GHz millimeter wave radiation on thecells 6 hours after exposure.

FIG. 5b illustrates the confluent cell culture exposed to 94 GHzmillimeter wave radiation and washed out 9 hours later to remove dead ordetached cells.

FIG. 6 illustrates applying millimeter wave radiation to two closelyadjacent regions.

FIG. 7 demonstrates sectioning of the cell culture by millimeter waveradiation.

DETAILED DESCRIPTION OF THE INVENTION

The subject application is directed to a device and method for selectivehyperthermic damage of target cells within the cells in a culturemedium, and more specifically, to a method and device for selectivehyperthermic damage of unwanted cells within a cell population withmillimeter wave radiation.

As used herein the term “culture medium” is used to refer to awater-based medium used to maintain live cells.

As used herein the term “cultureware” is used to refer to a containerused to hold cells placed in a culture medium.

As used herein the term “hyperthermic damage” is used to refer to damagedue to overheating.

As used herein the term “irreversible damage” relates to a degree ofdamage after which the cells could not survive.

Turning now to FIG. 1, there is shown a block diagram of a preferredembodiment of a device 100 for selective hyperthermic damage of targetcells in accordance with the subject application. As shown in FIG. 1,the device 100 includes a source 102 of millimeter wave radiation, anassociated cell cultureware 104 having cells in a culture medium 106placed therein, and a waveguide 108. The waveguide 108 is adapted fordelivering the millimeter wave radiation to a predetermined spot 110 ofan external surface 112 of the associated cell cultureware 104 toprovide for exposing a predetermined region 114 of exposure of the cellsin the cell culture medium 106 to the millimeter wave radiation. An endface 116 of the waveguide 108 is positioned in close proximity of thepredetermined spot 110.

In a preferred embodiment, the source 102 operates in the W-band, whichranges from 75 to 110 GHz. In other preferred embodiments, a source thatoperates in the F, D, or G-band could be used as the source 102.

A skilled artisan will appreciate that the source 102 comprises, forexample, and without limitation, a fixed frequency Gunn oscillator 118(InP or GaN Gunn diode) that produces a stable output signal in thefrequency range 75-110 GHz with a maximum power output of 65-100 mW, apassive variable attenuator 120 to control the radiation level, anarrow-band millimeter wave power amplifier 122 for increasing themillimeter wave radiation power to 250-400 mW or higher, power sources124, 126 to feed the Gunn oscillator 118 and the millimeter wave poweramplifier 122. As it will be appreciated by one skilled in the art, allabove elements of the source 102 are commercially available. All basicelements of the source 102 may be placed in a separate enclosure (notshown).

In the embodiment illustrated in FIG. 1, the predetermined spot 110 ofthe external surface 112 is located on a bottom 128 of the associatedcell cultureware 104. Thus the end face 116 of the waveguide 108 ispositioned below the bottom 128 of the associated cell cultureware 106.However, in another embodiment, the predetermined spot 110 may belocated on the top (lid) of the associated cell cultureware 104 (notshown in the drawing). Those skilled in the art will recognize that insuch an embodiment, the position of the waveguide 108 is adaptedaccordingly.

The internal dimensions of the waveguide 108 or any portions thereof aredetermined by the frequency of millimeter wave radiation. For example,for millimeter wave radiation of 94 GHz the internal dimensions of thewaveguide 108 are 2.540×1.270 mm (WR10), so the maximal diameter of theregion 114 of exposure determined by the smallest dimension of the endface of the waveguide 108 is around 1 mm.

Turning now to FIG. 2, there is shown a block diagram of a preferredembodiment of a device 200 for selective hyperthermic damage of targetcells in accordance with the subject application. As shown in FIG. 2,the device 200 includes a source 202 of millimeter wave radiation, anassociated cell cultureware 204 having cells in a culture medium 206placed therein, and a waveguide 208. The waveguide 208 is adapted fordelivering the millimeter wave radiation to a predetermined spot 210 ofan external surface 212 of the associated cell cultureware 204 toprovide for exposing a predetermined region 214 of exposure of the cellsin the culture medium 206 to the millimeter wave radiation. An end face216 of the waveguide 208 is positioned in close proximity of thepredetermined spot 210. Those skilled in the art will recognize that thesource 202 may be analogous to the source 102 of the device 100.

In the embodiment of FIG. 2, the device 200 for selective hyperthermicdamage of target cells further includes a visualizing module 218 adaptedfor visualizing the cells in the culture medium 206 placed in theassociated cell cultureware 204. As will be appreciated by those skilledin the art, the visualizing module 218 comprises a microscope 220,wherein the associated cell cultureware 204 having cells in the culturemedium 206 placed therein is positioned on a microscope stage 222. Thoseskilled in the art will further recognize that the microscope 220 iscapable of being implemented, for example, and without limitation, as afluorescent or a bright-field microscope, as an upright or an invertedmicroscope. In the embodiment of FIG. 2, the microscope 220 isimplemented as an upright microscope. In this embodiment, the end face216 of the waveguide 208 is positioned below a bottom 224 of theassociated cell cultureware 204 having cells in a culture medium 206placed therein, exposing thereby the predetermined region of exposure ofthe cells in the culture medium 206 located above the predetermined spot210, to the millimeter wave radiation.

Further, in the embodiment of FIG. 2, the waveguide 208 comprises afirst portion 226 positioned horizontally relative to the microscopestage 222 and a second portion 228 positioned upright relative to themicroscope stage 222, wherein the second portion 228 is connected withthe first portion 226 via a 90 degree E-plane waveguide bend 230. Aswill be understood by a skilled artisan, the portions of the waveguide208 are commercially available, wherein the 90 degree E-plane waveguidebend 230 may be custom made.

Those skilled in the art will recognize that the visualizing module 218may further comprise at least one of: a long working distance objective,a CCD camera or an infra-red camera for visualization on a monitor andimage capture, and a motorized stage to hold and move the associatedcell cultureware having cells in a culture medium placed therein (notshown in the drawings).

As will be further appreciated by those skilled in the art, the devicefor selective hyperthermic damage of target cells of the presentinvention may be implemented as a detachable device that can be easilyattached to any standard microscope (up-right or inverted) with minormodifications of the microscope.

Referring now to operation of the device for selective hyperthermicdamage of target cells 100 in accordance with the present invention,shown in FIG. 1, the operation of the device 100 commences by placingcells in the culture medium 106 within an associated cell cultureware104. The cells in the culture medium 106 are kept sterile by acultureware lid (not shown). Next, millimeter wave radiation from thesource 102 is delivered via the waveguide 108 to the predetermined spot110 of the external surface 112 of the associated cell cultureware 104having cells in the culture medium 106 placed therein. The radiationpower and exposure time of the millimeter wave radiation, and positionalrelationship of the end face 116 of the waveguide 108 and of the targetcells are selected such as to expose the predetermined region 114 ofexposure of the cells in the culture medium 106 to the millimeter waveradiation and provide selective hyperthermic damage to the target cellsin the predetermined region 114. The predetermined region of exposuremay have a diameter of less than 1.3 mm, preferably in the range of fromabout 0.25 mm to about 1.3 mm.

As will be understood by those skilled in the art, the millimeter waveradiation is absorbed in a very narrow layer (of about 0.4 mm or less)of the cells in the culture medium 106 and rapidly heats the narrowlayer of the cells in the culture medium 106 comprising the unwantedcells, without heating the bulk of the cell cultureware 104. The cellslocally heated by the millimeter wave radiation even for a short timeare damaged, if necessary, irreversibly damaged and eliminated.Preferably, the exposure time is about 2-3 sec, wherein the target cellsin the predetermined region 114 of exposure are heated to a temperatureof about 58-60° C.

Referring now to operation of the device for selective hyperthermicdamage of target cells 200 in accordance with the present invention,shown in FIG. 2, the operation of the device 200 commences by placingthe cells in the culture medium 206 within the associated cellcultureware 204, the latter being placed on the microscope stage 222.The cells in the culture medium 206 are kept sterile by a culturewarelid (not shown). Next, the millimeter wave radiation, for example at afrequency of 94 GHz, from the source 202 is delivered via the first 226and second 228 portions and the end face 216 of the waveguide 208 to thepredetermined spot 210 of the external surface 212 of the associatedcell cultureware 204 having cells in the culture medium 206 placedtherein. The radiation power and exposure time of the millimeter waveradiation, and positional relationship of the end face 216 of thewaveguide 208 and of the target cells are selected such as to expose thepredetermined region 214 of exposure of the cells in the culture medium206 to the millimeter wave radiation and provide selective hyperthermicdamage to the target cells in the predetermined region 214 of exposureof the cells in the culture medium 206.

Specifically, in the embodiment of FIG. 2, the millimeter wave radiationpasses through the first portion 226 of the waveguide 208 positionedhorizontally relative to the microscope stage 222, and is thenredirected by a 90 degree E-plane waveguide bend 230 mounted under themicroscope stage 222 of an up-right microscope 220 to the second portion228 positioned upright relative to the microscope stage 222. The cellsthat need to be selectively eliminated by irreversible hyperthermicdamage may be visualized within the culture medium 206 by means of avisualizing module 218.

For example, in a bright-field mode the cells may be illuminated througha condenser (not shown) placed below the waveguide 208. The waveguidebend 230 may comprise an orifice of ˜1 mm in diameter (not shown in thedrawing) in the lower surface of the waveguide bend 230 to allow for theillumination light to reach the cells through the second portion 228 ofthe waveguide 208. The light scattered by the cells is collected by anobjective (not shown in the drawing) focused on an internal surface ofthe cultureware 204. An image of the cells may be acquired by a CCDcamera (not shown) capable of maintaining the video rate for real timeimaging. The image or the real time stream may be digitized, displayedand stored by a computer (not shown). The microscope stage 222, ifmotorized, may be controlled by a joystick (not shown) and/or by thecomputer.

As known in the art, millimeter wave radiation is readily absorbed bywater. For W-band (75 to 110 GHz) the penetration depth is about 0.4 mmin water based media. The major fraction of the power of W-band enteringa water based medium decays exponentially within a layer 0.2-0.3 mmthick, i.e. the electromagnetic field energy is completely converted toheat in this narrow region.

Millimeter wave radiation can penetrate diamagnetic materials, such asglass and plastic, without power attenuation. When cells in the culturemedium 206 within the cultureware 204 are exposed to the millimeter waveradiation guided to the glass or plastic external surface 212 of thebottom 224 of the cultureware 204, the millimeter wave radiation heatsthe culture medium and the cells near the external surface 212 of thecultureware 204, but not the external surface 212 itself. Thetemperature of the culture medium near the external surface 212 dependson the energy (amplitude and frequency) of millimeter wave radiation.The bottom portion of the culture medium (within a bottom layer of <0.4mm thick) that is heated by millimeter wave radiation, moves up due tothe convection phenomena so there is no damage to the neighboring cellsaround the region 214 of exposure. The cells in the region 214 ofexposure are heated up to the temperature of about 60° C. that is muchhigher than the reported high-temperature survival threshold for allmammalian cells (48-50° C.).

The cells are irreversibly damaged in the region 214 of exposure whenthey are heated to about 60° C. within seconds. Heated (damaged) cellsdetach from bottom of the cultureware 204, become spherical andeventually are washed out when the media is changed.

Millimeter wave radiation is a form of non-ionizing radiation (meaningit can't directly break up atoms or molecules) that lies between commonradio and infrared frequencies. So it is not thought to damage DNA, theway X and gamma rays do. Nevertheless, millimeter wave radiation canobviously cause heating effects, and can harm or kill at high energies.In accordance with the subject invention, in order to heat cells up to60° C., about 100 mW of millimeter wave radiation power is applied to aregion with diameter ˜0.5-1 mm. A skilled artisan will appreciate thatthe millimeter wave radiation-mediated hyperthermic damage of thecurrent invention is safe for an operator because the cell mediacompletely absorbs all millimeter wave radiation power. Even if theequipment is accidently turned on without the cell cultureware 204 onthe stage 222, the millimeter wave radiation power is too low to harmthe operator. Even 100 mW millimeter wave radiation fanning out from theend face 216 of the waveguide 208 will expose the operator to much lessthan a maximum permissible exposure for uncontrolled environments of 10Wm⁻² (IEEE, 2005).

Further, to exclude a power-on without the cell cultureware 204 placedover the end face 216 of the waveguide 208 a simple millimeter waveradiation detector may be placed near the microscope objective (notshown in the drawings). The millimeter wave radiation detector may bearranged to “power off” the Gunn oscillator of the source 202, if highmillimeter wave radiation intensity is detected. One skilled in the artwill recognize that such detectors are well known in the art, and anysuch millimeter wave radiation detector is capable of being suitablyincluded in the device.

Below are Examples of using the method for selective hyperthermic damageof target cells in accordance with the present invention.

Calibration

Prior to operation, the device of the present invention is preferablycalibrated. The calibration procedure is illustrated in FIG. 3 and willbe described with reference to the embodiment shown in FIG. 2, though itshould be understood that a similar procedure may be performed for theembodiment of FIG. 1. The calibration procedure is performed to ensurethat the millimeter wave radiation level is high enough to heat thecells in the region of exposure to the desired temperatures (58-60° C.).

The calibration procedure starts by preparing a calibration plate 302that is similar to the cultureware 204. The internal surface 304 of thecalibration plate 302 is preferably coated with a thin film (about 10 μmthick) of long-chain alcohol (LCA) with a known melting pointtemperature. In a specific embodiment, the internal surface 304 iscoated with a thin film 306 of 1-Octadecanol (1-OD) that has a meltingpoint temperature of 58° C. Image A in FIG. 3 shows an unmelted 1-ODmicrofilm.

Next, a millimeter wave radiation at a frequency in the range of 75-110GHz with the time of exposure of about 2-3 seconds is applied to thecoated calibration plate 302 via a waveguide 308 with an end face 310positioned below the calibration plate 302. This leads to melting of anarea 312 in the 1-OD microfilm 306. The melting process may bevisualized with phase contrast microscopy: image A of FIG. 3 shows the1-OD microfilm 306 before application of the millimeter wave radiation,image B was acquired about 1 second after application of the millimeterwave radiation when the melted area 312 has started to appear, image Cwas acquired several seconds after application of the millimeter waveradiation when the melted area 312 has stopped extending and reached thesize of approximately 0.3 mm.

As will be appreciated by one skilled in the art, by adjusting exposureparameters such as the radiation power and exposure time of themillimeter wave radiation, and positional relationship of the end face310 of the waveguide 308 and of the calibration plate 302, a bigger orsmaller melting area 312 may be attained. Images D-E of FIG. 3illustrate obtaining a bigger melted area 322 of approximately 0.7 mm indiameter by increasing the power of the millimeter wave radiation (thearrow between images C and D indicates that the 1-OD microfilm 306 wasmoved to melt the bigger area 322 at a different position than theposition of the smaller area 312). In a specific embodiment, a 2× airobjective lens with a large working distance and a small numericalaperture (not shown in the drawing) was used to observe the microfilm306.

Lower millimeter wave radiation power results in a smaller diameter ofthe melting area 312. Thus, smaller melted microfilm area 312 may beobtained when the source 202 (FIG. 2) of millimeter wave radiation isadjusted to a lower power via a variable attenuator (not shown) and/orwhen the end face 310 of the waveguide 308 is positioned farther fromthe external surface 314 of the calibration plate bottom 316. Forprecise single cell hyperthermic damage the diameter of the region ofexposure may be decreased to 0.25 mm or less. The maximal size of themelting area 312 (about 1 mm in diameter) is determined by thewavelength of millimeter wave radiation in the W-band. Thus, theequipment can be calibrated to ablate cells in areas with differentdiameters.

For best results, the calibration procedure is preferably performedunder conditions very similar to the conditions at which the cells wouldbe treated: the calibration plate 302 with the microfilm containing thesame volume of the same culture medium as the cell cultureware 204 (FIG.2) with the cells placed therein. The LCA film 306 melts when theculture medium closely adjacent to the film 306 is heated viaapplication of the millimeter wave radiation. The heated culture medium318 moves upward creating a convectional flow 320; therefore the filmadjacent to the region of exposure is not heated (FIG. 3). As it wouldbe appreciated by one skilled in the art, in the absence of the culturemedium in the calibration plate 302, no melting of the LCA microfilm isobserved (data not shown). This is consistent with the fact that, asknown in the art, absorption of millimeter wave radiation by LCA, higheralkanes or oils is negligible.

Thus LCA/alcane microfilms may serve as local sensors of the temperaturein a narrow layer (˜10-15 μm thick) of cell culture medium at theglass/plastic bottom of the calibration plate 302. The LCA microfilm mayalso be formed directly in the cultureware 204 (FIG. 2) having cells ina culture medium 206 placed therein to allow performing the calibrationprocedure in the same plate, in close proximity to the cells.

After the millimeter wave radiation power is adjusted, the hyperthermicdamage process is started to expose the unwanted cells and/or cellcolonies to millimeter wave radiation. The hyperthermic damage resultsbecome visible in a few hours after the treatment. This time interval isshorter for higher temperatures or high millimeter wave radiationpowers. The cells exposed to lethal millimeter wave radiation powerdetach from the substrate and can be easily washed out by simple mediachange. If some unwanted cells are still found after the firsttreatment, the procedure can be repeated to completely eliminate them.

Effect of 94 GHz Irradiation (Heating to 60° C.) on U2OS CellsExpressing GFP-Tubulin.

As known in the art, mammalian cells do not survive even a short-termexposure to 60° C. To better define the time of irradiation required toirreversibly damage the cells in the region of exposure, the cellsexpressing GFP-tubulin were exposed to 94 GHz millimeter wave radiation.The power of millimeter wave radiation was adjusted to melt the 1-ODmicrofilm 306 (FIG. 3) with melting temperature ˜58° C. in 10-15seconds. Normally GFP-tubulin is excluded from the cell nucleus 402, 404(FIG. 4, panel A). It was observed, however, that after about 20 secondsof millimeter wave radiation GFP-tubulin started entering the cellnucleus and then the nuclear GFP-tubulin concentration equilibrated withthe cytoplasm 406, 408 (FIG. 4, panel B) indicating that the nuclearenvelope was compromised. This shows that a sudden disruption of thenuclear envelope permeability happens when cells are rapidly heated to58-60° C. as the proteins that are normally excluded from the nucleus(like GFP-tubulin) enter it.

The graph 400 in FIG. 4 shows the fluorescence intensity of the regionsof interest (dotted circles 410, 412) inside the nuclei of two cells asa function of time. The curve 414 on the graph shows the averageintensity in the circle 410 on panels A-B. The curve 416 on the graphshows the average fluorescence intensity in the circle 412 on panelsA-B. The sharp drop in the GFP fluorescence after application of 94 GHzmillimeter wave radiation (arrow at 3 seconds) is attributed to a dropin the GFP quantum yield when the temperature rises. The arrowheads markthe beginning of GFP-tubulin entry into the nucleus that indicatesdamage in the barrier function of the nuclear envelops.

It was also observed that cells exposed to the high temperaturesdemonstrate reversible membrane bleb formation (not shown).

Effect of 94 GHz Millimeter Wave Radiation on the Cells 6 Hours afterExposure.

FIG. 5a shows a follow up observation of a region 500 of exposure. Thefollow up observation of the region 500 was performed with a 20×objective using differential interference contrast (DIC). Theobservation of the region 500 performed 6 hours after the exposure tothe millimeter wave radiation revealed that the cells 502 in the region500 of exposure detach from the well bottom and become spherical.

Confluent Cell Culture Exposed to 94 GHz Millimeter Wave Radiation andWashed Out 9 Hours Later to Remove Dead or Detached Cells.

FIG. 5b shows a further follow up observation of the region 500 ofexposure. The next follow up observation of the region 500 was performedwith a 20× objective with a 20× objective using phase contrast. Theobservation performed 9 hours after the exposure to the millimeter waveradiation and after washing out the cells with a culture medium showedthat washing out the cells removes the spherical cells and leaves aclear (cell-free) internal surface surrounded by the confluent cellculture. The exposure parameters adjusted to obtain a desired diameterof the melting area in the calibration procedure described herein wereused to produce the same desired diameter of the region of exposure ofthe cells in the culture medium where the cells were totally eliminated.As seen from FIG. 5b , the neighboring cells 504 that grow at the edgeof the region 500 of exposure were not damaged. They continued to growand actively migrated to the now empty area (region 500).

Thus the method of the current invention allows for eliminating ofunwanted cells selectively even within confluent cell culture. Theelimination process may be repeated several times before the unwantedcells are completely removed.

Applying Millimeter Wave Radiation to Two Closely Adjacent Regions.

FIG. 6 illustrates applying of millimeter wave radiation to two closelyadjacent regions 602, 604 of exposure. As seen in FIG. 6, the cells inthese regions 602, 604 are completely eliminated via millimeter waveradiation irradiation. The two regions 602, 604 are in a very closeproximity to each other (˜250 μm). However, the cells between theregions 602, 604 survived the treatment and continue to grow. Image A isacquired using bright field phase contrast, image B depicts GFPfluorescence as the cells are expressing GFP-tubulin.

The method of the invention can be applied to cell suspensions as well.In this case prior to applying millimeter wave radiation it isnecessarily to wait until all the cells settle at the cultureware bottomand then mark the desired regions where the cells should survive. Theoperator (or software) can drive the microscope stage to expose cells inunmarked regions to millimeter wave radiation that heat them to 60° C.The heated cells will eventually die but the cells in the regions thatwere not exposed to millimeter wave radiation will survive and continueto grow.

Sectioning of the Cell Culture by Millimeter Wave Radiation.

The method of the invention may be used for making sections in aconfluent or semi-confluent cell culture. This may be done manually orwith the use of a software-driven motorized microscope stage, providedthat the stage is moved slow enough to expose the cells to lethalmillimeter wave radiation dose. The width of the sectioning line may be1 mm or less. The trajectory 702 of the sectioning line becomes visibleeven under low magnification bright-field objective (2×) because exposedcells round up and are observed as a light line (spherical dead cells)against surrounding live culture (flat and dark) as seen on FIG. 7 thatshows a bright field image of a confluent cell culture under 2×magnification. The light P-like line is a trajectory of the region ofexposure in the confluent cell culture (9 hours after treatment).

The following examples clarify possible applications of the describedmethod and device for selective hyperthermic damage of target cells.

Example 1

Selection of Desirable Single Cell Clones in Biotechnology Applications

In some applications cells are seeded at low density to isolate singlecell colonies for cell line development. Usually several cells areplaced per well in a cell culture plate (manually via delimited dilutionor automatically by flow cytometry sorting) to allow formation ofseparated colonies. If cells are single cell sorted or seeded to getsingle colonies, many cell lines show poor survival of single cellclones and slow initial outgrow rate. Better cell survival and growthrate are achieved with placement of several or more cells per well asthey condition the culture medium. As the end result, only onesingle-cell colony that possesses some useful properties (fluorescentprotein knock-in, the knock-out of a surface antigen, etc.) needs to beisolated. Therefore all unwanted colonies that helped the desired colonyto survive should be eliminated. Overheating by millimeter waveradiation offers a safe and effective way to do so. The elimination isbased on a feature distinguishing the desired cells that are kept alive(a specific morphology, a fluorescent signal coming from geneticallyengineered fluorescent proteins, a surface marker that could be revealedby fluorescently-labeled antibodies, etc.). The method of the currentinvention allows eliminating all unwanted colonies and keeping aliveonly a specific single cell colony that exhibits a desired phenotype.

Example 2

Generation of Tumor Cell Cultures Originated from Cancer Patient TumorBiopsy.

In another application, the tumor biopsies are collected from cancerpatients to establish the primary human tumor cell cultures. However,the biopsy samples are extremely heterogeneous due to the presence ofother tissue cells like fibroblasts. In most cases these cells grow fastin cultures and easily can overgrow the desired tumor cells. Thedisclosed device may be used to distinguish the unwanted contaminatingcells (via differences in morphology or using fluorescent labels) and toeliminate (ablate) them without damaging the neighboring tumor cells.The millimeter wave radiation hyperthermia ablation of fast-growingprimary non-tumor cells (fibroblasts or others) may provide superiorconditions for the growth of the desired tumor cells and may allowestablishing more homogenous culture of patient tumor cells.

Example 3

Coculture Micropatterning of Primary Cells.

Primary cells are thought to be the best test models of human organs andtissues for drug discovery and ADME/Tox profiling. The problem is thatthey are viable only for a few days and/or their organ-specificfunctions rapidly decline under conventional culture conditions.Coculture micropatterning of primary cells (e.g. hepatocytes) andsupporting cells (e.g. fibroblasts) creates primary cell clusteringknown to extend the lifespan of primary cells in culture and improvetheir organ-specific functions. The millimeter wave radiationhyperthermia ablation may be used to create micropatterned voids in thesupporting culture.

Then the voids can be seeded with the primary cells to createmicropatterned clusters surrounded by the supporting cells. Thisapproach provides a simple and inexpensive way to fabricatemicropatterned cocultures.

Example 4

Induced Pluripotent Stem (iPS) Cells and Embryonic Stem (ES) CellsTherapies.

Another application involves the use of induced pluripotent stem (iPS)cells to treat numerous human diseases. The iPS cells are capable ofdifferentiation in a way that is very similar to embryonic stem (ES)cells which can grow into fully differentiated tissues. iPS cells and EScells express the same cell surface antigenic markers. Both cell typesmight be used to generate different types of tissues via differentiationprocesses. Then the differentiated tissues (cardiomyocytes, hepatocytes,neurons, etc.) can be used in transplantation. However, differentiationof both iPS and ES cells usually yields mixed cell population i.e. bothdifferentiated and undifferentiated cells. The transplantation ofundifferentiated cells in patients might lead to tumor formation. Thus,the therapeutic use of tissues derived from both iPS and ES cells ispossible only after the purification i.e. complete elimination ofundifferentiated stem cells. The method described herein allowspurifying the mixed cell culture by selective elimination of practicallyall undifferentiated stem cells or cells that spontaneouslydifferentiated to another cell types. The purified tissue could beimplanted to the patients.

Example 5

Wound-Healing Assay.

The wound-healing assay is simple, inexpensive, and one of the earliestdeveloped methods to study directional cell migration in vitro. Thismethod mimics cell migration during wound healing in vivo. The basicsteps involve creating a “wound” in a cell monolayer, capturing theimages at the beginning and at regular intervals during cell migrationto close the wound, and comparing the images to quantify the migrationrate of the cells. It is particularly suitable for studies of theeffects of cell-matrix and cell-cell interactions on cell migration.Usually the wound is created my mechanical scratch of cell monolayer,via application of biocompatible hydrogel to bottom that creates acircular area across which cells may migrate following gel removal orvia placement of special silicone inserts to the cell culture surfacebefore cell seeding (when the insert is removed it creates well definedempty regions with no cells). The disclosed device could be used to makeprecise circular holes in cell monolayer without creating any mechanicalor chemical disturbance of the surrounding cells. The high temperature(60° C. or more) will induce the denaturation of all extracellularmatrix proteins that were produced by growing cells. So the migrationcells must produce them anew.

Example 6

Hyperthermia Therapy Studies

Hyperthermia therapy is a type of medical treatment in which body tissueis exposed to slightly higher temperatures to damage and kill cancercells or to make cancer cells more sensitive to the effects of radiationand certain anti-cancer drugs. Hyperthermia may kill or weaken tumorcells, and is controlled to limit effects on healthy cells. In manycases hyperthermia is performed via application of radiofrequency (RF)waves to ablate tumor tissues. Cancerous cells are not inherently moresusceptible to the effects of heat. When compared in in vitro studies,normal cells and cancer cells show the same responses to heat. However,the cancer cells might be selectively sensibilized to hyperthermiatreatment with special drugs. The disclosed device and method mayprovide an experimental tool for high-content screening of compoundsthat selectively sensibilize cancer cells to hyperthermia therapy. Forexample, the cancer cells might be mixed with normal cells to yield amixed cell culture. The tested compound is added to the culture mediaand after a pre-incubation period the millimeter wave radiation areapplied to the culture to increase the temperature to sub-lethal level(below 48° C.). If the tested compound makes cancer cells significantlyless able to tolerate the added heat stress than the healthy cells, themillimeter wave radiation application will lead to cancer cells dyingbut normal cells surviving. The found hit compounds could be used toincrease the effect of radiofrequency ablation in cancer patients.

Example 7

Generation of Single Cell Clones After DNA, mRNA or ProteinMicroinjection in Adherent Cells.

Novel genetically modified cell lines could be developed by variousmolecular biology methods for genome editing. An essential step in theprocess is the delivery of DNA, mRNA or protein (e.g. site specificnucleases) inside the parental cells. Microinjection is the most direct,predictable and effective way for the delivery but practically it isimpossible to isolate injected (and then genetically modified) cellsfrom the majority of the unmodified ones. The disclosed device may beused to visualize unmodified cells (via bright-field microscopy orfluorescent labels) and eliminate (ablate) them without damaging thedesired, genetically modified cells.

The foregoing description of preferred embodiments of the subjectapplication has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit the subjectapplication to the precise form disclosed. Obvious modifications orvariations are possible in light of the above teachings. The embodimentwas chosen and described to provide the best illustration of theprinciples of the subject application and its practical application tothereby enable one of ordinary skill in the art to use the subjectapplication in various embodiments and with various modifications as aresuited to the particular use contemplated. All such modifications andvariations are within the scope of the subject application as determinedby the appended claims when interpreted in accordance with the breadthto which they are fairly, legally and equitably entitled.

What is claimed:
 1. A device for selective hyperthermic damage of targetcells, comprising: a source of millimeter wave radiation; and awaveguide adapted for delivering the millimeter wave radiation to apredetermined spot of an external surface of an associated cellcultureware having cells in a culture medium placed therein, to providefor exposing a predetermined region of exposure of the cells in theculture medium to the millimeter wave radiation; wherein an end face ofthe waveguide is positioned in close proximity of the predeterminedspot, and wherein radiation power and exposure time of the millimeterwave radiation, and positional relationship of the end face of thewaveguide and the target cells are selected such as to provide selectivehyperthermic damage to the target cells in the predetermined region ofexposure of the cells in the culture medium, wherein the millimeter waveradiation is in a frequency band selected from W-band, F-band, D-band,or G-band, and has a water penetration depth of about 0.4 mm or less. 2.The device according to claim 1, further comprising a visualizing moduleadapted for visualizing the cells in the culture medium placed in theassociated cell cultureware.
 3. The device according to claim 2, whereinthe visualizing module comprises a microscope, wherein the associatedcell cultureware having the cells in the culture medium placed thereinis positioned on a microscope stage.
 4. The device according to claim 3,wherein the microscope is motorized.
 5. The device according to claim 3,wherein the microscope is a fluorescent or a bright-field microscope. 6.The device according to claim 3, wherein the microscope is an uprightmicroscope, and wherein the end face of the waveguide is positionedbelow a bottom of the associated cell cultureware having the cells inthe culture medium placed therein, exposing thereby the predeterminedregion of exposure of the cells in the culture medium located above thepredetermined spot, to the millimeter wave radiation.
 7. The deviceaccording to claim 6, wherein the waveguide comprises a first portionpositioned horizontally relative to the microscope stage and a secondportion positioned upright relative to the microscope stage, wherein thesecond portion is connected with the first portion via a 90 degreeE-plane waveguide bend.
 8. The device according to claim 3, wherein thevisualizing module further comprises at least one of: a long workingdistance objective, a CCD camera for image capture, an infra-red camerafor visualization on a monitor and a motorized stage to hold and movethe associated cell cultureware having the cells in the culture mediumplaced therein.